www.nature.com/scientificreports OPEN A novel metabarcoding diagnostic tool to explore protozoan haemoparasite diversity in Received: 20 June 2019 Accepted: 19 August 2019 mammals: a proof-of-concept study Published: xx xx xxxx using canines from the tropics Lucas G. Huggins 1, Anson V. Koehler1, Dinh Ng-Nguyen2, Stephen Wilcox3, Bettina Schunack4, Tawin Inpankaew5 & Rebecca J. Traub1 Haemoparasites are responsible for some of the most prevalent and debilitating canine illnesses across the globe, whilst also posing a signifcant zoonotic risk to humankind. Nowhere are the efects of such parasites more pronounced than in developing countries in the tropics where the abundance and diversity of ectoparasites that transmit these pathogens reaches its zenith. Here we describe the use of a novel next-generation sequencing (NGS) metabarcoding based approach to screen for a range of blood-borne apicomplexan and kinetoplastid parasites from populations of temple dogs in Bangkok, Thailand. Our methodology elucidated high rates of Hepatozoon canis and Babesia vogeli infection, whilst also being able to characterise co-infections. In addition, our approach was confrmed to be more sensitive than conventional endpoint PCR diagnostic methods. Two kinetoplastid infections were also detected, including one by Trypanosoma evansi, a pathogen that is rarely screened for in dogs and another by Parabodo caudatus, a poorly documented organism that has been previously reported inhabiting the urinary tract of a dog with haematuria. Such results demonstrate the power of NGS methodologies to unearth rare and unusual pathogens, especially in regions of the world where limited information on canine vector-borne haemoparasites exist. Protozoan haemoparasites generate some of the highest rates of morbidity and mortality in canines worldwide, whilst some are also zoonotic, capable of producing signifcant infections in humans as well1–4. Te principal taxonomic groups responsible are the bloodborne piroplasmids and kinetoplastids which are transmitted by haematophagous arthropods, such as ticks, feas, sand-fies and mosquitoes, as vector-borne diseases (VBDs)3,5. Examples of haemoparasite zoonoses include leishmaniasis which has long been identifed as an important canine VBD with a widespread, and in some regions expanding distribution6,7, whilst non-zoonotic diseases such as canine, equine or bovine babesiosis are nevertheless critically important diseases from a veterinary standpoint, with some species now recognised as key emerging pathogens8,9. Apicomplexan Babesia spp. parasites are transmitted by tick vectors which invade erythrocytes and cause a spectrum of anaemia-related pathology depending on the species, from the relatively benign Babesia vogeli to the more virulent Babesia canis and Babesia rossi species1,10. Whilst it has not been confrmed that canine-infecting Babesia spp. can infect people, other members of the genus, including Babesia microti present a severe zoonotic threat8,11. In the tropics, kinetoplastid parasites such as Trypanosoma evansi, that are important livestock path- ogens, can also frequently produce fatal infections in dogs4. Furthermore, canines are the primary zoonotic res- ervoir for Leishmania infantum, a kinetoplastid capable of causing a visceral, multi-organ disease in dogs and 1Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC, 3052, Australia. 2Faculty of Animal Sciences and Veterinary Medicine, Tay Nguyen University, Buon Ma Thuot, Dak Lak, 630000, Vietnam. 3Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia. 4Bayer Animal Health GmbH, Leverkusen, Germany. 5Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10900, Thailand. Correspondence and requests for materials should be addressed to L.G.H. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:12644 | https://doi.org/10.1038/s41598-019-49118-9 1 www.nature.com/scientificreports/ www.nature.com/scientificreports immunocompromised humans and children, particularly in regions of South America, the Middle East and the Mediterranean12–14. Many of these haemoparasites are united by their ability to create enduring infections, that can last years, with periods of immunological control followed by remission10,15,16. Tis tenet facilitates the formation of a haemopar- asite microbiome as a single host accumulates more infections, including those from bacteria, viruses and meta- zoans, some of which can be chronic and others short lived. Within the context of the canine blood microbiome a single haemoparasite may not be lethal, but still exert a toll on the dog in which it resides that may make the host more susceptible to other VBDs or make the pathogenesis of another parasite worse17,18. For instance, Hepatozoon canis, typically generates a subclinical infection15, however, when found to be coinfecting with Babesia spp. or bacterial VBDs a much more severe anaemia and overall disease outcome is generated18. Taking this into consid- eration, canine VBD diagnostic methods must be able to characterise the entire haemoparasite microbiome and not just a dominant pathogen within a host. With the advent of next-generation sequencing (NGS) technologies the feld of parasite diagnostic tests is in the process of being transformed. Conventional PCR (cPCR) methodologies such as endpoint or quantitative PCR, which themselves superseded laborious microscopy and culture-based methods, have always been lim- ited in their assessment of microbiomes by the need for a priori data on a target species taxonomic barcoding sequence19,20. Tis restrains such methods to only detecting known and genetically characterised species, whilst ignoring rare or undiscovered species19. Additionally, endpoint PCR coupled with Sanger sequencing is typically unable to detect more than the dominant sequence in an amplicon of potentially many, thereby making the technique of limited utility for recognising mixed infections21. NGS-based diagnosis mitigates such limitations as class, phylum or even kingdom specifc primers can be used to amplify around barcode regions that are unique to each species. Amplifed DNA from all the diferent species barcodes in a sample can be sequenced in massive parallelisation, generating a sample metabarcode of every species present from a taxonomic group of interest, thereby elucidating an entire microbiome from within a specifc host environment22,23. Te aim of this study was to develop an NGS-based diagnostic tool to fully characterise the apicomplexan and kinetoplastid haemoparasite microbiome from blood DNA samples, thereby including the ability to detect novel, rare or poorly documented species. Moreover, we aimed to compare this novel NGS-based method to the sensitivity and detection range of conventional PCR methods. Semi-domesticated and temple community canine populations from Tailand were chosen as there is relatively limited information regarding haemoparasite infec- tion in Southeast Asian dogs, whilst the few studies that have been conducted have found parasite prevalence to be high24–26. Results Design of metabarcoding primers for Apicomplexa and Kinetoplastida. Two primer pairs were designed to exclusively amplify 18S rRNA sequences from the phylum Apicomplexa and the class Kinetoplastida using a diverse range of sequences from GenBank. As the metabarcoding was to be carried out on canine blood samples, Apicomplexa and Kinetoplastida sequences from common blood-infecting species were chosen (for the complete list see the “Methods” section). Primers were designed to bind to highly conserved 18S rRNA sequences but fanking areas of high sequence diversity (barcode regions) to provide species-level discrimination. Host, canine 18S rRNA sequences were also included in the alignment to ensure that the designed primers did not cross-react with canine DNA. Metabarcoding assay validation. Confrmed haemoparasite positive controls were used to fnd optimal PCR conditions, whilst primer cross-reactivity was tested for, using positive controls from species outside of the taxa targeted by each primer pair (see the “Methods” section). Mock communities were also generated by mixing haemoparasite positive controls. Afer completion of our developed metabarcoding pipeline these mock commu- nities were consistently and accurately refected in the fnal results. Bioinformatic analysis of Apicomplexa data. In total 6,649,169 (median 62,120) raw paired-end reads were obtained for the 104 multiplexed Apicomplexa amplicons, including two positive and two negative controls. Afer the DADA2 quality fltering, dereplication, chimera removal and pair-joining step, a total of 564,332 joined sequences were retained, representing a retention of 16.97% of original reads (564,332 × 2 ÷ 6,649,169). NGS characterisation of Apicomplexa. Of the 100 canine blood samples tested 13 were found to be pos- itive for B. vogeli (mean reads 9,846; range 84–43,347) and 38 for H. canis (mean reads 8,774; range 90–58,207); six of these dogs were infected with both haemoparasite species. Two canine DNA samples returned sequences that could only be identifed to the level of phylum Apicomplexa (mean reads 120; range 119–121). When this sequence was run through a BLASTn search it returned a 100% identity result with a diverse range of sarcocysti- dae family pathogens, making species level assignment impossible with this sequence alone. In total 47% of dogs were found infected with at least one apicomplexan VBD via deep sequencing